1. Field of the Invention
The present invention relates to a time-of-flight/ion trap mass spectrometer, a method of operating the time-of-flight/ion trap mass spectrometer, and a computer program and a computer-program-product such as for example a hard drive or floppy disk or other medium containing computer code for operation of the time-of-flight/ion trap mass spectrometer.
2. Description of the Background
A mass spectrometer (MS) is used to determine the identity and quantity of constituent materials in a gaseous, liquid or solid specimen.
In a normal mode of operation, specimens are analyzed in mass spectrometers by ionizing the molecules of the specimen in an ion source, separating ions according to their mass-to-charge ratio (m/z) in a mass analyzer, and bombarding the separated ions in an ion detector to obtain a mass spectrum. Typically, the ion mass m is expressed in atomic mass units or Daltons (Da) and the ion charge z is the charge on the ion in terms of the number of elementary charges e.
In a tandem mode of operation, the mass spectrometer includes a device that produces fragmentation of ions into smaller structure-specific ions for further mass analysis. A spectrum referred to as a tandem mass spectrum corresponding to the fragmented ions can be obtained. By repeating the isolation and fragmentation stages a multiple number of times, one can obtain a tandem mass spectrum in this tandem mode of operation in which the ions have been repeatedly fragmented through a number of MS stages, e.g., a MSn spectra is obtained (where nxe2x89xa72) which thereafter are also referred to as tandem mass spectra. In mass analysis of large biological molecules, tandem mass spectral measurements provide structural and sequential information about peptides and other biopolymers.
A time-of-flight (TOF) mass spectrometer (MS) is a known instrument for mass analysis in a normal mode. In TOF-MS, ions formed from sample molecules in an ion source are accelerated to the same energy and allowed to drift along a defined path before detection. Because ions of different mass have different velocity, after acceleration, they are separated in space during flight and in time during detection. Thus, the time of arrival to the detector is measure of the mass or the mass-to-charge ratio m/z, if ions are not singly-charged.
This picture can be complicated by the presence of non-ideal factors, which include: (a) different time of formation or acceleration of ions; (b) different initial location of ions in space; and (c) different initial velocity of ions before acceleration. A number of methods, such as for example time focusing, dual-stage extraction, and time-lag focusing, can be used to correct these factors. Time focusing can be achieved by using pulsed drawout fields with sharp rise times or short laser pulses in the case of laser desorption (LD) or matrix-assisted laser desorption/ionization (MALDI). Alternatively, a dual-stage extraction method can be used to correct the initial spatial distribution of ions in an ion source. Initial velocity (or energy) distribution can be corrected by a time-lag focusing technique (e.g. a pulsed or delayed or time-delayed extraction method) or an ion mirror method (e.g., a method utilizing a reflectron). In addition, orthogonal ion extraction which interfaces with continuous ionization sources such as electrospray ionization (ESI), as described in U.S. Pat. No. 4,531,056, the entire contents of which are incorporated herein by reference, can be used to reduce the impact of the non-ideal factors.
Conventional TOF-MS schemes are shown in FIGS. 1A-1D which are discussed below. Details on a TOF-MS are given in: R. J. Cotter, Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research, ACS Professional Reference Books, Washington, D.C., 1997, pp. 1-327; W. C. Wiley, I. H. McLaren, Rev. Sci. Instr., 1955, vol. 26, pp. 1150-1157; A. F. Dodonov, I. V. Chernushevich, V. V. Laiko, in Time-of-Flight Mass Spectrometry, Ed. R. J. Cotter, American Chemical Society, Washington, D.C., 1994, pp. 108-123, the entire contents of each reference are incorporated herein by reference.
Time-of-flight (TOF) mass spectroscopy has several advantages over other types of mass spectroscopy. A TOF mass spectrometer is conservative of the sample since every ion formed in the bunch is detected. Open flight tube designs in TOF mass spectrometers result in high ion transmittance due to a wide aperture to the source. There is no fundamental limit (other than detectability) on the range of analyzed m/z values. Due to the pulsed nature of the TOF technique, the TOF mass spectrometer can be interfaced to pulsed ion sources; importantly, it can be interfaced, as shown in FIGS. 1A-1C, to vacuum MALDI ion sources which are widely utilized for the ionization of large biological molecules as described in M. Karas, F. Hillenkamp, Anal. Chem. 1988, vol. 60, pp. 2299-2301, the entire contents of which are incorporated herein by reference, and the afore-mentioned electrospray ionization sources.
FIG. 1A shows a linear TOF-MS in which a laser 100 desorbs an ionized species from a sample contained in a matrix-assisted laser desorption/ionization stage 102. The stage 102 exists at a high voltage potential adjacent to an extraction device 104. Ionized species are extracted from the region near the stage 102 and directed to an ion detector 106.
FIG. 1B shows a reflection TOF-MS similar to the TOF-MS in FIG. 1A, but where ions are directed by reflectron 108 to the ion detector 106 through a curved path. The reflection 108 includes a series of rings with each ring set progressively to higher potentials.
FIG. 1C shows a TOF-MS in which a pulsed voltage signal is applied to the stage 102 after the laser 100 produces the desorbed, ionized species. The pulse voltage signal time-focuses the desorbed ionized species to ensure that ions of a particular m/z value arrive at the ion detector simultaneously. Most commercial MALDI instruments use a TOF-MS as a mass analyzer.
Further, U.S. Pat. No. 5,965,884, the entire contents of which are incorporated herein by reference, describes an atmospheric pressure MALDI technique in which ions are formed outside the vacuum of a mass spectrometer at atmospheric conditions. FIG. 1D shows an orthogonal acceleration TOF-MS interfaced with an atmospheric ion source. Ions from the atmospheric ion source 110 pass through a heated capillary 112 and a skimmer 114 to produce a collimated ion beam. Once inside the vacuum of the orthogonal acceleration TOF-MS, electrostatic optics 116 focus the ion beam into a quadrupole 118 which homogenizes the ion beam such that ions exiting the quadrupole 118 have only an axial velocity component and almost no radial velocity. Deflection optics 120 deflect the exiting ions toward the reflectron 108 which in turn reflects the ions to the ion detector 106.
However, a drawback of TOF-MS technology is that TOF-MS does not easily provide for a tandem mode of operation. Yet, tandem experiments, by analysis of fragmentation patterns from complex molecules, can play a role in structural elucidation of biological molecules. This role of TOF-MS for MALDI analysis of biological molecules has led to the search for mechanisms to provide a tandem mode of operation in TOF-MS instruments.
In the tandem mode, the isolated ions (i.e., precursor ions) undergo an activation process to produce fragmented ions. Activation energy of the precursor ions can come from collisions with buffer gas or surfaces or photoexcitation. One approach, as shown in FIG. 2A and as described in U.S. Pat. No. 5,202,563, the entire contents of which are incorporated herein by reference, utilizes a tandem reflectron TOF instrument (reTOF/reTOF) with a collision chamber for providing activation and fragmentation. FIG. 2A shows a tandem reflectron mass spectrometer in which ions transit through two reflectrons 220 and 222 and a collision chamber 202 before arriving at the ion detector 106.
In another approach, as shown in FIG. 2B and as described in B. Spengler, D. Kirsch, R. Kaufmarn, E. Jaeger, Rapid Commun. Mass Spectrom., 1992, vol. 6, pp. 105-108, the entire contents of which are incorporated herein by reference, a postsource decay (PSD) process fragments the ionized species. The fragmentation occurs as a part of the natural decay process via the residual energy remaining in the ionized species from the laser desorption/ionization event. FIG. 2B shows that ions extracted from the stage 102 pass through an ion gate 204 selecting ions of interest. The ion gate passes ions of interest which fragment due to PSD phenomenon before entering the reflectron 220, enabling subsequent separation and analysis of fragment ions at the ion detection 106.
In other approaches, product ions from the ion source are re-accelerated using two linear TOF mass analyzers (i.e., without reflectrons), as described in D. R. Jardine, J. Morgan, D. S. Alderdice, P. J. Derrick, Org. Mass Spectrom., 1992, vol. 27, pp. 1077-1083 and K. L. Schey, R. G. Cooks, R. Grix, H. Wollnik, Int. J Mass Spectrom. Ion Proc., 1987, vol. 77, pp. 49-61, the entire contents of each reference are incorporated herein by reference. Further, a linear/reflectron (TOF/RTOF) configuration has been described in K. L. Schey, R. G. Cooks, A. Kraft, R. Grix, H. Wollnik, Int. J Mass Spectrom. Ion Proc., 1989, vol. 94, pp. 1-14, the entire contents of which are incorporated herein by reference.
Further, a hybrid sector/reflectron TOF instrument utilizing a double-focusing sector mass analyzer for mass selection and a reflectron TOF to record the product ions was used, as described in F. H. Strobel, T. Solouki, M. A. White, D. H. Russell, J Am. Soc. Mass Spectrom., 1990, vol. 2, pp. 91-94 and in F. H. Strobel, L. M. Preston, K. S. Washburn, D. H. Russell, Anal. Chem., 1992, vol. 64, pp. 754-762, the entire contents of which are incorporated herein by reference.
In a reflectron TOF (reTOF) mass analyzer, a focusing problem exists. Before fragmentation, product ions in the mass spectrometer have the same velocity as the precursor ions, but after fragmentation, the kinetic energy of the fragmented product ions differs from that of the precursor ions so that the reflectron which is a monochromatic device, i.e. a device designed to focus ions at one energy, does not focus all the fragmented ions. In one corrective approach, reflectron voltages are stepped to record different regions of the mass spectrum (e.g., scanning of the reflectron voltages), and a focused mass spectrum is reconstructed from a series of transient spectra, as described in R. Weinkauf, K. Walter, C. Weickhardt, U. Boesl, E. W. Schlag, Int. J. Mass Spectrom. Ion Processes, 1989, vol. 44a, pp. 1219-1225, the entire contents of which are incorporated herein by reference and in B. Spengler, D. Kirsch, R. Kaufmann, E. Jaeger, Rapid Commun. Mass Spectrom., 1992, vol. 6, pp. 105-108, the entire contents of which has been previously incorporated herein by reference. Alternatively, a curved field reflectron as described in U.S. Pat. No. 5,464,985, the entire contents of which are incorporated by reference, improves the mass resolution for fragmented product ions.
An ion trap, as shown in FIG. 2C, has also been utilized as a front-end device on TOF-MS instruments. These instruments, referred to as IT/TOF-MS, are described in S. M. Michael, B. M. Chien, D. M. Lubman, Rev. Sci. Instrum., 1992, vol. 63, 4277; T. L. Grebner, H. J. Neusser, Int. J Mass Spectrom. Ion Proc., 1994, vol. 137, Li; Q. Ji, P. R. Vlasak, M. R. Davenport, C. G. Enke, J. F. Holland, J Am. Soc. Mass Spectrom., 1996, vol. 7, 1009, and V. M. Doroshenko, R. J. Cotter, J Mass Spectrom., 1998, vol. 33, pp. 305-318, the entire contents of each reference are herein incorporated by reference.
An electrodynamic ion trap is a device consisting of several electrodes in which ions can be trapped in space for a long time by applying periodic or alternating potentials to one or several electrodes to generate an inhomogeneous-in-space trapping electric field. The ion trap (IT) of FIG. 2C consists of one annular ring electrode 206 and two ion-trap end-cap electrodes 208 and 210. A RF voltage is applied to the ring electrode 206 while the ion-trap end-cap electrodes 208 and 210 are grounded during most of the operational cycle time. The electric field inside the ion trap typically includes a main quadrupole field as well as higher order, weaker fields. Such an ion trap is commonly referred to as a quadrupole ion trap. Ions from an ion source are accumulated in the trap, fragmented inside the trap using normal ion trapping procedures (e.g., by resonant excitation and collisional activation), and extracted for mass analysis. The extracted ions (as shown in FIG. 2C) transit a curved path to the ion detector 106 by passing through a reflectron 220.
In contrast to a vast majority of tandem mass spectrometers typically called xe2x80x9ctandem-in-spacexe2x80x9d mass spectrometers which include additional mass analyzers for isolating and analyzing ions, ion isolation and mass analyzing in an ion trap can be performed within the same volume of the mass spectrometer. For this reason, an ion trap is often referred to as a xe2x80x9ctandem-in-timexe2x80x9d mass spectrometer. Details on ion trap design and the operation of an ion trap as a mass spectrometer are described in: R. E. March, R. J. Hughes, Quadrupole Storage Mass Spectrometry, John Wiley and Sons, NY, N.Y., 1989, pp. 1-471; R. G. Cooks, G. L. Glish, S. A. McLuckey, R. E. Kaiser, xe2x80x9cIon Trap Mass Spectrometryxe2x80x9d, CandEN, 1991, March 25, pp. 26-41; and German Patent No. 944,900 and U.S. Pat. Nos. 2,939,952; 3,065,640; 4,540,884; 4,882,484; 5,107,109; and 5,714,755, the entire contents of each reference are incorporated herein by reference.
One advantage of ion-trap mass spectrometry is the ability of the ion-trap to perform tandem mass spectrometry. However, only a limited mass range is available due to a low-mass cut-off phenomenon at the low mass end and a decreasing trapping well depth at the high mass end. Also, mass assignment accuracy in a IT-mass spectrometer is compromised by space charge effects when there is a high population of the trapped ions in the ion trap. A variety of ionization sources have been interfaced with ion traps including MALDI sources, as described in V. M. Doroshenko, T. J. Cornish, R. J. Cotter, Rapid Commun. Mass Spectrom., 1992, vol. 6, 753-757, the entire contents of which are incorporated herein by reference. Despite an effective method for injection of MALDI ions into the trap whereby RF voltages are ramped while ions are injected into the trap as described in U.S. Pat. No. 5,399,857, the entire contents of which are incorporated herein by reference, a MALDI ion trap mass spectrometer is still not available commercially due to the limited mass range of current ion trap devices.
Another related ion trapping method, applicable when injecting short duration ion beams, involves the application of pulsed retarding DC voltages to one of the ion trap electrodes for deceleration of injected ions as described in J. E. Crowford, F. Buchinger, L. Davey, Y. Ji, J. K. P. Lee, J. Pinard, J. L. Vialle, W. Z. Zhao, Hyperfine Interact., 1993, vol. 81, p. 143, the entire contents of which are incorporated herein by reference.
In another approach, as shown in FIG. 2D, multiple quadrupoles as described in Chernushevich et al., Rapid Commun. Mass Spectrom., 1997, vol. 11, pp. 1015-1024, the entire contents of which are incorporated by reference, were utilized in conjunction with an orthogonal extraction TOF instrument (i.e., a qqTOF configuration) to achieve a tandem mode of operation. FIG. 2D shows that a quadrupole section 212a provides ion isolation, a quadrupole section 212b provides fragmentation, and a quadrupole section 212c provides thermalization. However, only a limited mass range is realized in the qq-TOF configuration.
As discussed above, conventional time-of-flight mass spectroscopy fails to address the requirement for tandem operation needed for structural elucidation of biological molecules. Previous attempts to achieve tandem operation in TOF-MS are complicated (as in the TOF/TOF technique), limited in mass resolution, mass range, and/or sensitivity, or merely simulate a true tandem operation (as in the PSD process).
One object of this invention is to provide a method and apparatus for tandem mode of operation in time-of-flight mass spectrometry in such a way that the time-of-flight mass spectrometer of the present invention has high mass range, high mass accuracy for the mass assignment, and high mass resolution.
Another object of the present invention is to provide a novel mass spectrometer which combines high mass range and high mass resolution performance of TOF-MS with the tandem capabilities of an ion trap mass spectrometer.
Since a sharing of an ion source between a free-standing TOF-MS and a free-standing IT-MS is complicated by the requirements for high precision ion optics, another object of the present invention is to provide an integrated TOF-IT mass spectrometer in which an ion extraction device directs ions to one of a TOF or an IT mass analyzer. The TOF-IT mass spectrometer of the present invention configures an extraction device which directs extracted ions into the TOF mass analyzer in a normal mode and directs extracted ions into an IT mass analyzer in a tandem mode. As a result, the extraction device operates in both normal and tandem modes with performance uncompromised by the presence of an ion trap mass analyzer in the system.
These and other objects are provided for in a mass spectrometer which includes an ion source, an extraction device, a TOF mass analyzer, an ion trap mass analyzer, and an ion guiding optical element which guides at least one of extracted ions from the ion source and extracted ion fragments into the TOF mass analyzer in a normal mode of operation and into the IT mass analyzer in a tandem mode of operation. The apparatus operates by producing ions from a sample, extracting the ions from the ion source, selecting between the TOF mass analyzer and the IT mass analyzer, directing extracted ions to the selected mass analyzer, mass-separating the directed ions and fragments of the directed ions according to a mass-to-charge ratio, detecting mass-separated ions with the selected mass analyzer, and producing at least one of a normal mass spectrum and a tandem mass spectrum.
In the spectrometer of the present invention, ions generated from an ion source can be mass analyzed independently by two mass spectrometers in a normal or tandem mode of operation. In the normal mode, the mass spectrometer of the present invention utilizes time-of-flight spectroscopy to survey a broad range of atomic mass units (AMU) extracted from the ion source, such as for example 1-100,000 AMU. In the tandem mode, the mass spectrometer of the present invention utilizes ion-trap spectroscopy to promote fragmentation of predetermined mass ranges within the broad spectrum of extracted ions. For example, a biological sample shows in a normal mode a peak around 1,500 AMU attributed to a peptide ion from a family of proteins. Subsequent fragmentation of the peptide ion would yield mass spectra unique to a specific protein in that family. Thus, in this example, the mass spectrometer of the present invention is utilized to obtain broad spectral analysis and fragmentation of selected ions to yield protein identification.
The method of the present invention is advantageous for analyzing ions generated from matrix assisted laser desorption/ionization (MALDI) ion sources and electrospray ionization sources which produce intact ions of biomolecules in a broad mass range.